The PITX1 gene is an important transcription factor implicated in both skeletal variation and disease. Predominantly expressed in the lower limbs and pelvis, PITX1 has recently been linked to height variation in human populations, suggesting that regulatory changes contribute to common traits. PITX1 has also been implicated as causal in clubfoot disease, a serious musculoskeletal abnormality with an estimated prevalence of one per 1000 live births. While there are documented coding changes in PITX1 in familial clubfoot, protein- coding changes have not been found in isolated cases. However, right-sided predominance of sporadic clubfoot is similar to right-biased anatomical changes documented in PITX1 mutant mice and stickleback fish, suggesting that many additional clubfoot cases may be due to unrecognized regulatory mutations in this gene. Yet despite the importance of PITX1 in human height and pelvic disease, its regulatory landscape is not well understood. Fortunately, work in the stickleback fish model system has now identified not one, but two, distinct pelvic enhancers in the PITX1 gene as underlying much of the variation in the pelvic girdle and pelvic spine. As these structures are homologous to the mammalian pelvis and hindlimbs, the sticklebacks are an ideal system for further studying natural PITX1-mediated pelvic variation. Using the stickleback PITX1 gene as a model, the precise regulatory mechanisms controlling a gene that is pivotal in mammalian development will be dissected. The Specific Aims of this proposal are to: (1) Determine the effect of targeted pelvic enhancer deletion in stickleback fish, (2) Identify naturally occurring variants in PITX1 pelvic enhancers, and (3) Determine regulatory potential of enhancer variants found in natural populations. These Aims will employ a variety of methods, including targeted genome editing in stickleback fish, comparative genomics in vertebrate species, and identification of human pelvic enhancer variants relevant to height and clubfoot disease, and functional testing of different enhancers in both in vivo and in vitro systems. In this proposal, we will test the contributions that each enhancer makes to the overall development of pelvic structures in stickleback fish. The generality of these mechanisms will be mechanisms will be further explored. By better understanding the regulatory landscape around PITX1, it will be possible to better understand, diagnose, and eventually treat cases of misregulation of this gene, such as idiopathic clubfoot. Identifying and characterizing limb enhancer elements regulating this gene will provide an important advance to the field, as physicians would know where to do targeted sequencing of patients, or, for whole genome sequencing, would be able to quickly narrow down the possible causative mutations.